Abstract Cocaine addiction involves the loss of control over drug taking so that individuals take more drug over time and can have prolonged vulnerability to relapsing to drug seeking, even after extended periods of abstinence. The progressive molecular and synaptic adaptations in neurons in the CNS that underlie these changes are not well understood, but can be studied using animal models based on extended cocaine self-administration followed by abstinence in rats. These models have shown that the nucleus accumbens core (NAcC), a small but critical brain region in ventral striatum, is implicated in compulsive cocaine taking and relapse to cocaine seeking after extended abstinence. The NAcC receives and integrates afferent information from many different brain regions and has two main output projections, the direct and indirect pathways; these pathways tend to oppose one another functional, and we predict that adaptations in signaling processes within these neurons are critical determinants affecting the relapse to drug seeking. By understanding the adaptations in cell signaling in these NAcC output neurons following extensive cocaine exposure and abstinence, we hope to contribute to novel treatment strategies for reducing the potential for relapse to drug seeking. We propose to investigate the time-dependent increase in drug seeking during abstinence known as the ?incubation of craving?. We will use several innovative tools. First, we will use an intersectional viral vector approach to introduce DREADDs and other transgenic proteins to perturb and study direct and indirect pathway neurons selectively during incubation. By injecting AAV vectors with floxed and inverted transgenes into NAcC, we can activate transgene expression selectively in the direct or indirect pathway neurons by injecting the ventral tegmental area or ventral pallidum with CAV2-Cre, which is retrogradely transported to the cell bodies in NAcC. Second, we will use engineered ?DREADD? receptors, a technology that we helped to establish for use in rat brain during complex behavioral experiments. DREADDs will allow us to activate Gs or Gi signaling pathways selectively in either direct or indirect pathway neurons during either repeatedly during cocaine taking or during early or late forced abstinence, thereby assessing how these canonical second messenger pathways modulate the plasticity involved in escalation or incubation. Third, we will utilize RiboTag technology to immunopurify polyribosomes selectively from direct or indirect pathway neurons and investigate the changes in RNA translation in these opposing pathways during abstinence and incubation of craving, both in cell bodies and in the synapses where activity dependent changes in local protein translation has been described. By perturbing and measuring signaling pathways in specified neurons, we hope to develop new strategies for ameliorating the adaptations associated with compulsive drug use and relapse to seeking.